The present invention relates to methods and compositions for inhibiting cell proliferative disorders. The described methods are particularly useful for inhibiting cell proliferative disorders characterized by over-activity and/or inappropriate activity of a receptor tyrosine kinase.
Receptor tyrosine kinases belong to a family of transmembrane proteins and have been implicated in cellular signaling pathways. The predominant biological activity of some receptor tyrosine kinases is the stimulation of cell growth and proliferation, while other receptor tyrosine kinases are involved in arresting growth and promoting differentiation. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed. (Schlessinger, J. and Ullrich, A., Neuron, 9(3):383-391, 1992.)
Receptor tyrosine kinases contain at least seven structural variants. All of the receptor tyrosine kinases are composed of at least three domains: an extracellular glycosylated ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self-phosphorylation (autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues.
Receptor phosphorylation stimulates a physical association of the activated receptor with target molecules. Some of the target molecules are in turn phosphorylated, which transmits the signal to the cytoplasm. For example, phosphorylation of phospholipase C-xcex3 activates this target molecule to hydrolyze phosphatidylinositol 4,5-bisphosphate, generating two secondary signal transducing molecules: inositol triphosphate, which causes release of stored intracellular calcium, and diacylglycerol, which is the endogenous activator of a serine/threonine kinase, protein kinase C. Other target molecules are not phosphorylated, but assist in signal transmission by acting as docking or adapter molecules for secondary signal transducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the Grb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras (reviewed in Schlessinger, J. and Ullrich, A., Neuron, supra).
The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, S. A., Science, 254:1146-1153, 1991; Schlessinger, J. Trends Biochem. Sci., 13:443-447, 1988; and Ullrich, A., and Schlessinger, J., Cell, 61:203-212, 1990.
Various cell proliferative disorders have been associated with defects in different signaling pathways mediated by receptor tyrosine kinases. According to Aaronson, S. A., supra:
Signaling pathways that mediate the normal functions of growth factors are commonly subverted in cancer.
Examples of specific receptor tyrosine kinases associated with cell proliferative disorders include, platelet derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and HER2. The gene encoding HER2 (her-2) is also referred to as neu, and c-erbB-2 (Slamon, D. J., et al., Science, 235:177-182, 1987).
HER2/neu gene amplification has been linked by some investigators to neoplastic transformation. For example Slamon et al., supra, (hereby incorporated by reference herein) asserts:
The Her-2/neu oncogene is a member of the erB-like oncogene family, and is related to but distinct from the epidermal growth factor receptor. The gene has been shown to be amplified in human breast cancer cells.
According to Scott et al., supra, (hereby incorporated by reference herein):
Amplification and/or overexpression of HER2/neu has been detected in gastrointestinal, non-small cell lung, and ovarian adenocarcinomas and occurs in a significant fraction of primary human breast cancers where it correlates with regionally advanced disease, increased probability of tumor recurrence, and reduced patient survival. (Citations omitted).
Publications discussing EGFR and cancer include Zeillinger et al., Clin. Biochem. 26:221-227, 1993; where it is asserted:
Increased expression of this receptor [EGFR] has been found in various malignancies. In carcinomas of the cervix, ovaries, esophagus, and stomach, positive EGF-R status is definitely associated with the aggressiveness of the tumor.
With regard to breast cancer the importance attached to the determination of EGF-R has been confirmed by reports by several groups on the positive correlation between EGF-R and relapse-free interval, as well as overall survival. (Citations omitted.)
Other references discussing cancer and EGFR include Karameris et al., Path. Res. Pract. 189:133-137, 1993; Hale et al., J. Clin. Pathol 46:149-153, 1993; Caraglia et al., Cancer Immunol Immunother 37:150-156, 1993; and Koenders et al., Breast Cancer Research and Treatment 25:21-27, 1993). (These references, which are not admitted to be prior art, are hereby incorporated by reference herein.)
Compounds able to inhibit the activity of receptor tyrosine kinases have been mentioned in various publications. For example, Gazit et al., J. Med. Chem. 34:1896-1907 (1991), examined the receptor tyrosine kinase inhibitory effect of different tyrphostins. According to Gazit:
Among the novel tyrphostins examined we found inhibitors which discriminate between the highly homologous EGF receptor kinase (HER1) and ErbB2/neu kinase (HER2). These findings may lead to selective tyrosine kinase blockers for the treatment of diseases in which ErbB2/neu is involved.
In a later publication Gazit et al., J. Med. Chem. 6:3556-3564 (1993) (not admitted to be prior art) describe tyrphostins having a S-aryl substituent in the 5 position. According to Gazit:
We find that these compounds are potent blockers of EGFR kinase and its homolog HER-2 kinase. Interestingly, we find that certain S-aryltyrphostins discriminate between EGFR and HER-2 kinase in favor of the HER-2 kinase domain by almost 2 orders of magnitude. When examined in intact cells it was found that these selective S-aryltyrphostins are equipotent in inhibiting EGF dependent proliferation of NIH 3T3 harboring either the EGF receptor or the chimera EGF/neu HER1-2.
OSherov et al., Journal of Biological Chemistry 268:11134, 1993 (not admitted to be prior art), mentions the development of two groups of tyrphostins:
one is highly selective in inhibiting HER1 [EGF] as compared with HER2 kinase activity, and the other is highly selective in inhibiting HER2 activity compared with HER1 kinase activity.
The present invention concerns methods and compounds which can be used to inhibit EGFR and/or HER2 activity, preferably HER2 activity. The described methods and compositions are particularly useful for treating cell proliferative disorders, such as cancers characterized by over-activity or inappropriate activity of HER2 or EGFR.
Groups of compounds able to inhibit HER2, and groups of compounds able to inhibit EGFR are described herein (See, FIG. 1). Also described, are exemplary compounds belonging to different groups, and guidelines which can be used to obtain additional compounds belonging to the different groups.
In addition to use as a therapeutic additional uses of the described compounds including use for in vitro studies to determine the mechanism of action of receptor tyrosine kinases, preferably HER2 or EGFR; use as lead compounds to design and screen for additional compounds able to effect receptor tyrosine kinase activity, preferably inhibit HER2 or EGFR, activity; and use to help diagnose the role of a receptor tyrosine kinase in a cell proliferative disorder. For example using standard assays, the active site of the kinase acted upon by any one of the compounds described herein may be determined, and other compounds active at the same site determined.
xe2x80x9cCell proliferative disordersxe2x80x9d refer to disorders wherein unwanted cell proliferation of one or more subset(s) of cells in a multicellular organism occurs, resulting in harm (e.g., discomfort or decreased life expectancy) to the multicellular organism. Cell proliferative disorders can occur in different types of animals and in humans. Cell proliferative disorders include cancers, blood vessel proliferative disorders, and fibrotic disorders.
A particular disorder is considered to be xe2x80x9cdrivenxe2x80x9d or caused by a particular receptor tyrosine kinase if the disorder is characterized by over-activity, or inappropriate activity, of the kinase and a compound which can inhibit one or more receptor tyrosine kinase activities exerts a therapeutic effect when administered to a patient having the disorder.
A xe2x80x9ctherapeutic effectxe2x80x9d generally refers to either the inhibition, to some extent, of growth of cells causing or contributing to a cell proliferative disorder; or the inhibition, to some extent, of the production of factors (e.g., growth factors) causing or contributing to a cell proliferative disorder. A therapeutic effect relieves to some extent one or more of the symptoms of a cell proliferative disorder. In reference to the treatment of a cancer, a therapeutic effect refers to one or more of the following: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell infiltration into peripheral organs; 3) inhibition (i.e., slowing to some extent, preferably stopping) of tumor metastasis; 4) inhibition, to some extent, of tumor growth; and/or 5) relieving to some extent one or more of the symptoms associated with the disorder. In reference to the treatment of a cell proliferative disorder other than a cancer, a therapeutic effect refers to either: 1) the inhibition, to some extent, of the growth of cells causing the disorder; 2) the inhibition, to some extent, of the production of factors (e.g., growth factors) causing the disorder; and/or 3) relieving to some extent one or more of the symptoms associated with the disorder.
When used as a therapeutic the compounds described herein are preferably administered with a physiologically acceptable carrier. A physiologically acceptable carrier is a formulation to which the compound can be added to dissolve it or otherwise facilitate its administration. Examples of physiologically acceptable carriers include water, saline, physiologically buffered saline, cyclodextrins and PBTE:D5W (described below). Hydrophobic compounds are preferably administered using a carrier such as PBTE:D5W. An important factor in choosing an appropriate physiologically acceptable carrier is choosing a carrier in which the compound remains active or the combination of the carrier and the compound produces an active compound. The compound may also be administered in a continuous fashion using a slow release formulation or a pump to maintain a constant or varying drug level in a patient.
Thus, a first aspect of the present environment describes a class of receptor tyrosine kinase inhibitor compositions. By an xe2x80x9cinhibitorxe2x80x9d of a receptor tyrosine kinase is meant that the compound reduces to some extent one or more activities of HER2, EGFR and/or PDGFR. Preferably, tyrosine kinase inhibitors can significantly inhibit the activity of HER2, EGFR and/or PDGFR. By xe2x80x9csignificantly inhibitxe2x80x9d is meant the compound has an IC50 less than 50 xcexcM in an assay described in the examples below. The compositions are made up of a compound having the chemical formula: 
where R1, R2, and R3 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, thioether, SH, halogen, hydrogen, NO2 and NH2; and R5 is an alkylaryl comprising an alkyl group, and an aryl group having the following structure: 
where X1, X2, X3, X4, and X5 is each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, OH, trihalomethyl, and NO2; preferably hydrogen, halogen, alkyl, trihalomethyl, and NO2.
Another aspect of the present invention describes a second class of receptor tyrosine kinase inhibitor compositions. These compositions are made up a compound having the chemical formula: 
where R1, R2, and R3 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, thioether, SH, halogen, hydrogen, NO2 and NH2;
Y is either, nothing, xe2x80x94C(CN)xe2x95x90Cxe2x80x94, -alkyl-, xe2x80x94NH-alkyl-; and
R5 is either CN or aryl.
Another aspect of the present invention describes a third class of receptor tyrosine kinase inhibitor compositions. These compositions are made up of a compound having the chemical formula: 
where R1, R2, R3 and R6 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, halogen, hydrogen, OH, NO2, amine, thioether, SH and NH2; and
X1, X2, X3, X4, and X5 are each independently selected from the group consisting of hydrogen, halogen, trihalomethyl, and NO2, provided that at least one of X1, X2, X3, X4, and X5 is a trihalomethyl.
Another aspect of the present invention describes a fourth class of receptor tyrosine kinase compositions. These compositions are made up of a compound having the chemical formula: 
where R1 and R3 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl; and
R4 is selected from the group consisting of alkyl, alkylaryl, amide, and thioamide.
Another aspect of the present invention describes a fifth class of receptor tyrosine kinase compositions. These compositions are made up of a compound having the chemical formula: 
where R7, R8, R9, and R10, is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, NO2, amine, thioether, SH, halogen, hydrogen and NH2;
R12 has the chemical structure: 
xe2x80x83where X6 is either O or S and X7 is either methyl or trihalomethyl; and
R13 is either aryl or alkylaryl.
Another aspect of the present invention describes a compound able to inhibit protein tyrosine kinase activity selected from the group consisting of: M16, N17, N21, N22, N23, N29, R9, R10, R11, R12 and R13.
Another aspect of the present invention a method of treating a patient having a cell proliferative disorder characterized by over-activity or inappropriate activity of a receptor tyrosine kinase, preferably EGFR, PDGFR, or HER2, more preferably HER2. The method involves the step of administering to a patient a therapeutically effective amount of one the compounds described herein. Preferably, the cell proliferative disorder is a cancer.
Another aspect of the present invention describes a method of treating a patient having a cell proliferative disorder characterized by inappropriate or over-activity of HER2. The method involves administering to the patient a therapeutically effective amount of a compound able to significantly inhibit HER2 activity. Preferably, the compound selectively inhibits HER2. The composition is selected from one of the following:
a) a compound having the chemical formula: 
xe2x80x83where R1, R2, R3, and R6 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, NO2, amine, thioether, SH, halogen, hydrogen and NH2; and
R4 is selected from the group consisting of alkyl, alkylaryl, thioamide, amide, CN, and sulfonyl.
b) a compound having the chemical formula: 
xe2x80x83where R7, R8, R9, and R10, is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, NO2, amine, thioether SH, halogen, hydrogen and NH2;
R12 is selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, ester, amide, thioamide, alkylaryl, trihalomethyl, CN, OH, amine, thioether, SH, NH2, and hydrogen; and
R13 is selected from the group consisting of aryl, alkyl, alkenyl, alkynyl, CN, alkylaryl, amide, and thioamide;
c) a compound having the chemical formula: 
xe2x80x83where R15, R16, R17, R18 and R19, is each independently selected from the group consisting of hydrogen alkyl, alkenyl, alkynyl, alkoxy, OH, NO2, amine, thioether, and SH; and
R20 is selected from the group consisting of alkyl, aryl, and alkylaryl; and
d) a compound having the chemical formula: 
xe2x80x83where R21, R22, R23, R24, and R25, are each independently selected from the group consisting of hydrogen, halogen, OH, SH, alkyl, aryl, trihaloalkyl, preferably hydrogen, halogen, OH, or SH;
R26 is either CH2 or NH;
R27 is either aryl or xe2x95x90C(CN)2; and
R28 is either nothing or H, provided that if R28 is nothing a double bond is present between N and R27.
e) compound R9, R11, R13, and R15.
Different types of cell proliferative disorders characterized by inappropriate or over-activity of HER2 can be treated using the compounds and methods described herein. Examples of such disorders include: cancers such as blood cancers; breast carcinomas, stomach adenocarcinomas, salivary gland adenocarcinomas, endometrial cancers, ovarian adenocarcinomas, gastric cancers, colorectal cancers, and glioblastomas, where the cancer is characterized by over-activity or inappropriate activity of HER2.
Another aspect of the present invention describes a method of treating a patient having a cell proliferative disorder characterized by inappropriate EGFR activity. The method involves administering to the patient a therapeutically effective amount of a compound able to significantly inhibit EGFR activity. Preferably, the compound selectively inhibits EGFR. Several EGFR inhibitor compounds fall within the generic structures of HER2 compounds described in the previous aspects. Additionally, compounds R10 and R11 can selectively inhibit EGFR activity.
Another aspect of the present invention describes a method of determining the importance of a receptor tyrosine kinase in cellular growth. The method involves the steps of:
a) contacting a cell with a composition comprising a compound which significantly inhibits the activity of one or more receptor tyrosine kinases selected from the group consisting of: EGF-R, PDGF-R, and HER2; and
b) measuring cell growth after step (a). Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.